Establishment Of Premature Ovarian Failure In Animals By Environmental Chemical VCD

BackgroundThe etiology and mechanism of Premature ovarian failure(POF) are complicated,and there're no reasonable and effective methods to cure.POF would affect patient's life quality seriously and lead to certain diseases directly.For example,premature ovarian failure may lead to infertility,ahead of menostasia and osteoporosis.So,to further discuss the etiology of POF and to search an effective method is the key point to solve the problem.In view of the low feasibility of study in human body,establishing a POF model similar to human's reproductive endocrinology and histology changes is very important to investigate the mechanism and therapy of POF.The females are born with a finite pool of primordial follicles that cannot be regenerated, the vast majority are lost through a degradative process called atresia,less than 1 percent primordial follicles will mature and ovulate. As a result,exposure to chemicals that cause depletion of the primordial follicles can jeopardize the reproductive capacity of females and may cause early menopause.There're reports that environmental chemicals can destroy the oocyte of follicles and affect the ovary function.Because people are exposed to ever-increasing combinations of environmental chemicals, occupational exposure may increase the risk of infertility and POF. So,there is an increased awareness of environmental chemicals'ovotoxity.4-vinylcyclohexene diepoxide(VCD) is one of the main metabolic products of 4-vinylcyclohexene(VCH), VCH is a product of rubber, flame retardants, pesticide, plastics and antioxidant production.there're reports that VCH and VCD can selectively destroy the primordial and primary follicles of the mice and Macaque. it was determined that VCD is the ovotoxic form of the parent compound VCH, Consequently, POF model study have utilized VCD. By observing vaginal smear,ovarian histology changes in different time points to evaluate the model.The aim is to observe the ovarian failure gradually,investigate the achievement ratio of the model and to judge the ability of simulating physiology POF,serving as a method and foundation for the study of POF etiology and therapy.Objective1. To establish the mice model of POF by using enviromental chemical VCD.2. To observe and check the relevant markers of ovarian function by VCD.3. To observe the process of ovarian failure gradually.4. To show more attention to environment agent.Materials and Methods1. Experimental subjects:The experimental subjects were 60 female kunming mice,28 days old,weighted from 80-100 grams,purchased from the animal experiement center of Shandong university, The room temperature was maintained at 20-24℃and the relative humidity is 50%-60%, Animals were given food and water ad libitum, and maintained on a 12-h light/dark cycle.2. Experimental methods2.1 Experimental schedule60 Kunming mice were divided into four groups at random,including VCD group, adjuvant control group,normal group and castrated group, VCD group and adjuvant control group were treated daily with VCD or vehicle at the same dose 5ml/kg i.p separately.vaginal smear is done 2 weeks after dosing to observe the estrous cycle changes.Meanwhile, Ovaries and uterus were removed and processed for histological evaluation at different time points after the onset of dosing and follicle counts are also performed to observe the morphology and histology changes of the uterus and ovaries.2.2 Grouping and therapy: A (VCD group):20 mice, treated daily with VCD 160mg/kg (5ml/kg),15 days, i.p separately.B (adjuvant control group,sesame oil group):16 mice, treated daily with sesame oil 5ml/kg, 15 days, i.p separately.C (normal group):20 mice,not be treated.D (castrated group):4 mice,ovaries were removed on the first day of injecting.2.3 The outcome measurement2.3.1 Common conditions observing the mice's activity,diet and weight changes after treating.2.3.2 Estrous cycles:vaginal discharging smear and HE dyeing to observe the estrous cycle changes.2.3.3 Ovaries and uterus'histologic changes:Ovaries and uterus were removed and processed for histological evaluation at different time points(on day18,37,80 and 120) after the onset of dosing, the slice thickness is 5 um, we got one slice every 20 um.2.3.4 Follicle counts at different time points:Follicles were counted on day 18,37,80,120 after the onset of dosing.To avoid double counting,primordial and primary follicles were counted when we saw the oocyte neclei,and secondary and antral follicles'oocyte nucleolus. We counted 20 sections per ovary.Results1. Estrus cycles changes:Eighty percent of VCD group mice displayed disruptions in estrous cyclicity on day 60,and all treated mice were in disturbance on day 80.there's a stable estrous cycles of sesame oil and normal group mice and all in diestrus of castrated group.2. Follicle counts and histology changes of the ovary:On day 18 following onset of treatment, primordial follicles of VCD group disappeared,primary follicles is decreased while larger follicles were unaffected compared with controls; On day 37, primary follicles disappeared or seen by chance and secondary and antral follicles were reduced to 10.08%(8.33/82.67) of normal group,the total number of follicles is 3.96%(11/278) of normal group;On day 80,there was a substantial reduction in the number of secondary and antral follicles (P<0.01);On day 120,the growing follicles disappeared or seen by chance,the ovaries and uterus showed apparently atrophy(5/5),cotex thickened and structure disturbance. There're kinds of follicles in different stages in normal and sesame oil group.3. The mophology and histology changes of the uterus:On day 120,VCD group showed uterus atrophy,endomembrane thinned and gland dicreased,similar to the castrated group.while in normal and sesame oil group,there'er thicker endomembrane and plentiful gland in lamina propria.Conclusion1. Anminal model of POF can be successfully established by using environmental chemical VCD, the experimental method is simple and achievement ratio is high.2. This study showed that VCD. can selectively destroy the primordial and primary follicles,and finally induced the secondary and antral follicles decreased or disappeared, suggested that the target of VCD may be primodial and primary follicles,the histologic observing showed no inflammation or necrosis,suggesting the mechanism of POF model is through apoptosis.3. The POF model by using VCD is similar to female physiological POF process,which is useful to clinical study and therapy of POF.4. To protect female humans from ovotoxity chemicals is of vital importance to female's reproductive health.